Rickettsia monteiroi sp. nov., infecting the tick Amblyomma incisum in Brazil

Free-living adult Amblyomma incisum ticks were collected in an Atlantic rainforest area at Intervales State Park, State of São Paulo, Brazil. From an A. incisum specimen, rickettsiae were successfully isolated in Vero cell culture by the shell vial technique. Rickettsial isolation was confirmed by optical microscopy, transmission electron microscopy, and PCRs targeting portions of the rickettsial genes gltA, htrA, rrs, and sca1 on infected cells. Fragments of 1,089, 457, 1,362, and 443 nucleotides of the gltA, htrA, rrs, and sca1 genes, respectively, were sequenced. By BLAST analysis, the partial sequence of rrs of the A. incisum rickettsial isolate was closest to the corresponding sequence of Rickettsia bellii (99.1% similarity). The gltA partial sequence was closest to the corresponding sequences of “Candidatus Rickettsia tarasevichiae” (96.1% similarity) and Rickettsia canadensis (95.8% similarity). The htrA partial sequence was closest to the corresponding sequence of R. canadensis (89.8% similarity). The sca1 partial sequence was closest to the corresponding sequence of R. canadensis (95.2% similarity). Since our rickettsial isolate was genetically distinct from other Rickettsia species, we propose a new species designated Rickettsia monteiroi sp. nov. Phylogenetic analyses indicated that R. monteiroi belongs to the canadensis group within the genus Rickettsia, together with the species R. canadensis and “Candidatus R. tarasevichiae”. Little or no antibody cross-reaction was observed between sera of R. monteiroi-inoculated guinea pigs and R. bellii-, Rickettsia rickettsii-, or R. canadensis-inoculated guinea pigs.     

Diversity of Rickettsiales in the Microbiome of the Lone Star Tick,Amblyomma americanum

Ticks are important vectors for many emerging pathogens. However, they are also infected with many symbionts and commensals, often competing for the same niches. In this paper, we characterize the microbiome of Amblyomma americanum (Acari: Ixodidae), the lone star tick, in order to better understand the evolutionary relationships between pathogens and nonpathogens. Multitag pyrosequencing of prokaryotic 16S rRNA genes (16S rRNA) was performed on 20 lone star ticks (including males, females, and nymphs). Pyrosequencing of the rickettsial sca0 gene (also known as ompA or rompA) was performed on six ticks. Female ticks had less diverse microbiomes than males and nymphs, with greater population densities of Rickettsiales. The most common members of Rickettsiales were “Candidatus Rickettsia amblyommii” and “Candidatus Midichloria mitochondrii.” “Ca. Rickettsia amblyommii” was 2.6-fold more common in females than males, and there was no sequence diversity in the sca0 gene. These results are consistent with a predominantly vertical transmission pattern for “Ca. Rickettsia amblyommii.”     

Wide Dispersal and Possible Multiple Origins of Low-Copy-Number Plasmids in Rickettsia Species Associated with Blood-Feeding Arthropods

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, “Candidatus Rickettsia amblyommii,” R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two “Ca. Rickettsia amblyommii” isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of “Ca. Rickettsia amblyommii,” R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.The alphaproteobacteria of the genus Rickettsia (Rickettsiales; Rickettsiaceae) have undergone the reductive genome evolution typical of obligate intracellular bacteria, resulting in A/T-rich genomes (1.1 × 10⁶ to 1.5 × 10⁶ bp) with a high content of pseudogenes undergoing elimination (3, 10, 20, 26). Initial sequencing of rickettsial genomes focused on the important arthropod-borne pathogens Rickettsia prowazekii, Rickettsia conorii, and Rickettsia typhi and appeared to confirm the prevailing belief that plasmids were absent and transposons were rare among Rickettsia spp. (2, 28, 39, 44). As mobile genetic elements in bacteria, plasmids and transposons drive horizontal gene transfer (HGT) and the acquisition of virulence determinants and environmental adaptive traits (30, 43, 60, 70). Subsequent sequencing of the Rickettsia felis genome revealed the surprising presence of abundant transposase paralogs and the 63-kbp pRF plasmid, with 68 open reading frames (ORFs) encoding predicted proteins, as well as a 39-kbp deletion form, pRFδ (45). Although pRF was suggested to be conjugative, it was initially thought to be unique among the rickettsiae, a reasonable inference given that plasmids are uncommon among the reduced genomes of obligate intracellular bacteria and were previously unknown in the Rickettsiales (3, 4, 13). However, a phylogenetic analysis implied an origin for pRF in ancestral rickettsiae and the possible existence of other rickettsial plasmids (28), which was soon confirmed by the cloning of the 23.5-kbp pRM plasmid from Rickettsia monacensis (6). Some of the 23 ORFs on pRM had close pRF homologs, and both plasmids carried transposon genes and the molecular footprints of transposition events associated with HGT from other bacterial taxa.The discoveries of pRF and pRM made obsolete the long-held dogma that plasmids were not present in members of the genus Rickettsia and implied a source of unexpected genetic diversity in the reduced rickettsial genomes, particularly if potentially conjugative plasmids carrying transposon genes proved to be common among members of the genus. That hypothesis gained credence when pulsed-field gel electrophoresis (PFGE) and Southern blot surveys (7) using plasmid gene-specific probes demonstrated plasmids in Rickettsia helvetica, “Candidatus Rickettsia hoogstraalii” (38), and Rickettsia massiliae and possible multiple plasmids in “Candidatus Rickettsia amblyommii” (71) isolates. The same study demonstrated the loss of a plasmid in the nonpathogenic species Rickettsia peacockii during long-term serial passage in cultured cells and the absence of a plasmid in Rickettsia montanensis M5/6, an isolate with a long laboratory passage history. Genome sequencing of R. massiliae and Rickettsia africae revealed the 15.3-kbp pRMA and 12.4-kbp pRAF sequences, with 12 and 11 ORFs, respectively, that were more similar to those of pRF than to those of pRM (11, 24).The absence of plasmids in R. montanensis and important Rickettsia pathogens maintained as laboratory isolates has left unresolved the question of the true extent of plasmid distribution among Rickettsia spp. Until recently, the genus was thought to consist of closely related species, known chiefly as typhus and spotted fever pathogens transmitted by lice, fleas, mites, and ticks (31). It is now apparent that many, and possibly most, Rickettsia spp. inhabit a diverse range of arthropods that do not feed on blood, as well as leeches, helminths, crustaceans, and protozoans, suggesting an ancient and complex evolutionary history (54). A multigene phylogenetic analysis of the Rickettsiales resulted in a “molecular clock” which indicated that the order arose from a presumably free-living ancestor and then adapted to intracellular growth during the appearance of metazoan phyla in the Cambrian explosion (76). A transition to a primary association with arthropods followed during the Ordovician and Silurian periods. The genus Rickettsia arose approximately 150 million years ago and evolved into several clades, including the early-diverging hydra and torix lineages associated with leeches and protozoans. A rapid radiation occurred about 50 million years ago in the arthropod-associated lineages (76).Whole-genome sequencing has led to a revision of phylogenetic relationships among Rickettsia spp. associated with blood-feeding arthropods (10, 26, 28). A newly defined ancestral group (AG) contains the earliest-diverging species, Rickettsia bellii and Rickettsia canadensis, while R. prowazekii and R. typhi, transmitted by lice and fleas, respectively, constitute the typhus group (TG). A proposed transitional group (TRG), consisting of the mite-borne Rickettsia akari, the flea-borne R. felis, and the tick-borne Rickettsia australis, bridges the genotypic and phenotypic differences between the TG and the much larger spotted fever group (SFG), consisting of tick-borne rickettsiae (28). However, some presumptive SFG rickettsiae remain poorly characterized and are of uncertain phylogenetic status, while the accumulation of genomic data from rickettsiae found in a diverse range of invertebrate hosts may have profound impacts on the currently understood phylogeny of rickettsiae associated with blood-feeding arthropods. For example, it appears that the above AG and TRG species have many close relatives in insects (76). Despite the recent phylogenomic advances, the genetic and host-adaptive mechanisms underlying the evolution of arthropod-transmitted pathogens of vertebrates from ancestral Rickettsia spp., including any possible role of plasmids, remain poorly understood.In this report, we have taken advantage of recent isolations of rickettsiae from North and South America to conclusively demonstrate that low-copy-number plasmids are indeed common in low-passage isolates of AG, TRG, and SFG rickettsiae. The only exceptions were multiple isolates of R. parkeri, obtained from ticks and human eschar biopsy specimens and newly recognized as a mildly pathogenic SFG rickettsia (49, 50, 52, 79), and the previously characterized species R. montanensis (7). We confirmed that some Rickettsia isolates harbor more than one plasmid by cloning and sequencing multiple plasmids from “Ca. Rickettsia amblyommii” isolates AaR/SC and Ac/Pa, and we obtained PCR- and gel-based evidence that supported genome sequence evidence for the existence of multiple plasmids in REIS, the rickettsial endosymbiont of Ixodes scapularis. Phylogenetic analysis provided strong evidence for multiple plasmid incompatibility groups and possible multiple origins of plasmid-carried parA genes in the genus Rickettsia. Other than genes encoding plasmid replication initiation and partitioning proteins, the newly sequenced “Ca. Rickettsia amblyommii” plasmids resembled the previously sequenced rickettsial plasmids in sharing limited similarities in coding capacity (6, 7, 22). However, we have previously drawn attention to the presence of hsp genes, encoding α-crystalline small heat shock proteins, as a conserved feature of most rickettsial plasmids that may play a role in host adaptation (7). Phylogenetic analysis indicated that the hsp2 genes were plasmid specific, while the hsp1 genes found on four rickettsial plasmids may have been acquired by a chromosome-to-plasmid transfer event in a TRG-like species.     

Evolutionary Relationships of Three New Species of Enterobacteriaceae Living as Symbionts of Aphids and Other Insects

Ecological studies on three bacterial lineages symbiotic in aphids have shown that they impose a variety of effects on their hosts, including resistance to parasitoids and tolerance to heat stress. Phylogenetic analyses of partial sequences of gyrB and recA are consistent with previous analyses limited to 16S rRNA gene sequences and yield improved confidence of the evolutionary relationships of these symbionts. All three symbionts are in the Enterobacteriaceae. One of the symbionts, here given the provisional designation “Candidatus Serratia symbiotica,” is a Serratia species that has acquired a symbiotic lifestyle. The other two symbionts, here designated “Candidatus Hamiltonella defensa” and “Candidatus Regiella insecticola,” are sister groups to one another and together show a relationship to species of Photorhabdus.     

“Candidatus Accumulibacter” Population Structure in Enhanced Biological Phosphorus Removal Sludges as Revealed by Polyphosphate Kinase Genes

We investigated the fine-scale population structure of the “Candidatus Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of “Candidatus Accumulibacter” 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “Candidatus Accumulibacter” lineage. Sequences from at least five clades of “Candidatus Accumulibacter” were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using “Candidatus Accumulibacter”-specific 16S rRNA and “Candidatus Accumulibacter” clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “Candidatus Accumulibacter” lineage and the relative distributions and abundances of the five “Candidatus Accumulibacter” clades. The qPCR-based estimation of the total “Candidatus Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined “Candidatus Accumulibacter” clades. The relative distributions of “Candidatus Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “Candidatus Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.     

Diversity of “Candidatus Liberibacter asiaticus,” Based on the omp Gene Sequence

Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, “Candidatus Liberibacter africanus” in Africa and “Candidatus Liberibacter asiaticus” in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of “Ca. Liberibacter asiaticus” isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species “Ca. Liberibacter asiaticus,” even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating “Ca. Liberibacter asiaticus” isolates.     

Discovery of Novel Rickettsiella spp. in Ixodid Ticks from Western Canada

The genomic DNA from four species of ixodid ticks in western Canada was tested for the presence of Rickettsiella by PCR analyses targeting the 16S rRNA gene. Eighty-eight percent of the Ixodes angustus (n = 270), 43% of the I. sculptus (n = 61), and 4% of the I. kingi (n = 93) individuals examined were PCR positive for Rickettsiella, whereas there was no evidence for the presence of Rickettsiella in Dermacentor andersoni (n = 45). Three different single-strand conformation polymorphism profiles of the 16S rRNA gene were detected among amplicons derived from Rickettsiella-positive ticks, each corresponding to a different sequence type. Furthermore, each sequence type was associated with a different tick species. Phylogenetic analyses of sequence data of the 16S rRNA gene and three other genes (rpsA, gidA, and sucB) revealed that all three sequence types were placed in a clade that contained species and pathotypes of the genus Rickettsiella. The bacterium in I. kingi represented the sister taxon to the Rickettsiella in I. sculptus, and both formed a clade with Rickettsiella grylli from crickets (Gryllus bimaculatus) and “R. ixodidis” from I. woodi. In contrast, the Rickettsiella in I. angustus was not a member of this clade but was placed external to the clade comprising the pathotypes of R. popilliae. The results indicate the existence of at least two new species of Rickettsiella: one in I. angustus and another in I. kingi and I. sculptus. However, the Rickettsiella strains in I. kingi and I. sculptus may also represent different species because each had unique sequences for all four genes.     

Molecular Evidence for the Broad Distribution of Anaerobic Ammonium-Oxidizing Bacteria in Freshwater and Marine Sediments

Previously available primer sets for detecting anaerobic ammonium-oxidizing (anammox) bacteria are inefficient, resulting in a very limited database of such sequences, which limits knowledge of their ecology. To overcome this limitation, we designed a new primer set that was 100% specific in the recovery of ~700-bp 16S rRNA gene sequences with >96% homology to the “Candidatus Scalindua” group of anammox bacteria, and we detected this group at all sites studied, including a variety of freshwater and marine sediments and permafrost soil. A second primer set was designed that exhibited greater efficiency than previous primers in recovering full-length (1,380-bp) sequences related to “Ca. Scalindua,” “Candidatus Brocadia,” and “Candidatus Kuenenia.” This study provides evidence for the widespread distribution of anammox bacteria in that it detected closely related anammox 16S rRNA gene sequences in 11 geographically and biogeochemically diverse freshwater and marine sediments.     

Genome sequence of Rickettsia bellii illuminates the role of amoebae in gene exchanges between intracellular pathogens

The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair–long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic “melting pot” where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.     

Enrichment and Genome Sequence of the Group I.1a Ammonia-Oxidizing Archaeon “Ca. Nitrosotenuis uzonensis” Representing a Clade Globally Distributed in Thermal Habitats

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called “marine” group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as “Candidatus Nitrosotenuis uzonensis”, is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F₄₂₀ and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, “Candidatus N. uzonensis” also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in “Ca. N. uzonensis”, which potentially include genetic determinants of ecological niche differentiation.     

Diverse Phage-Encoded Toxins in a Protective Insect Endosymbiont

The lysogenic bacteriophage APSE infects “Candidatus Hamiltonella defensa,” a facultative endosymbiont of aphids and other sap-feeding insects. This endosymbiont has established a beneficial association with aphids, increasing survivorship following attack by parasitoid wasps. Although APSE and “Ca. Hamiltonella defensa” are effectively maternally transmitted between aphid generations, they can also be horizontally transferred among insect hosts, which results in genetically distinct “Ca. Hamiltonella defensa” strains infecting the same aphid species and sporadic distributions of both APSE and “Ca. Hamiltonella defensa” among hosts. Aphids infected only with “Ca. Hamiltonella defensa” have significantly less protection than those infected with both “Ca. Hamiltonella defensa” and APSE. This protection has been proposed to be connected to eukaryote-targeted toxins previously discovered in the genomes of two characterized APSE strains. In this study, we have sequenced partial genomes from seven additional APSE strains to address the evolution and extent of toxin variation in this phage. The APSE lysis region has been a hot spot for nonhomologous recombination of novel virulence cassettes. We identified four new toxins from three protein families, Shiga-like toxin, cytolethal distending toxin, and YD-repeat toxins. These recombination events have also resulted in reassortment of the downstream lysozyme and holin genes. Analysis of the conserved APSE genes flanking the variable toxin cassettes reveals a close phylogenetic association with phage sequences from two other facultative endosymbionts of insects. Thus, phage may act as a conduit for ongoing gene exchange among heritable endosymbionts.     

Genomic Features of a Bumble Bee Symbiont Reflect Its Host Environment

Here, we report the genome of one gammaproteobacterial member of the gut microbiota, for which we propose the name “Candidatus Schmidhempelia bombi,” that was inadvertently sequenced alongside the genome of its host, the bumble bee, Bombus impatiens. This symbiont is a member of the recently described bacterial order Orbales, which has been collected from the guts of diverse insect species; however, “Ca. Schmidhempelia” has been identified exclusively with bumble bees. Metabolic reconstruction reveals that “Ca. Schmidhempelia” lacks many genes for a functioning NADH dehydrogenase I, all genes for the high-oxygen cytochrome o, and most genes in the tricarboxylic acid (TCA) cycle. “Ca. Schmidhempelia” has retained NADH dehydrogenase II, the low-oxygen specific cytochrome bd, anaerobic nitrate respiration, mixed-acid fermentation pathways, and citrate fermentation, which may be important for survival in low-oxygen or anaerobic environments found in the bee hindgut. Additionally, a type 6 secretion system, a Flp pilus, and many antibiotic/multidrug transporters suggest complex interactions with its host and other gut commensals or pathogens. This genome has signatures of reduction (2.0 megabase pairs) and rearrangement, as previously observed for genomes of host-associated bacteria. A survey of wild and laboratory B. impatiens revealed that “Ca. Schmidhempelia” is present in 90% of individuals and, therefore, may provide benefits to its host.     

Infection by <Emphasis Type="Italic">Rickettsia bellii</Emphasis> and <Emphasis Type="Italic">Candidatus “Rickettsia amblyommii”</Emphasis> in <Emphasis Type="Italic">Amblyomma neumanni</Emphasis> Ticks from Argentina

The tick species, Amblyomma neumanni (Acari: Ixodidae) is the most frequent tick parasitizing humans in northwestern Argentina. The present study evaluated the rickettsial infection among 55 A. neumanni adult free-living ticks collected in Dean Funes, Córdoba Province. Ticks were individually processed by the hemolymph test with Gimenez staining, isolation of rickettsia in Vero cell culture by the shell vial technique, and polymerase chain reaction (PCR) targeting the citrate synthase rickettsial gene. Through the shell vial technique, rickettsiae were successfully isolated and established in Vero cell culture from two ticks (ticks 4 and 13), which previously showed to contain Rickettsia-like organisms by the hemolymph test. These two Rickettsia isolates were designated as An4 and An13. Molecular characterization (partial DNA sequences of two to three rickettsial genes were determined) of these two isolates and phylogenetic analyses identified them as Rickettsia bellii (isolate An4) and Candidatus “Rickettsia amblyommii” (isolate An13). After testing all A. neumanni ticks by PCR, the prevalence of Candidatus R. amblyommii and R. bellii was 23.6% (13/55) and 3.6% (2/55), respectively. These two rickettsiae have been considered of unknown pathogenicity and appropriate studies to test their pathogenicity to humans or animals need to be conducted. This is the first report of Rickettsia in ticks from Argentina, and also in the species A. neumanni. The results reinforce previous findings that R. bellii (and probably Candidatus R. amblyommii) are widespread among some Neotropical Amblyomma species, suggesting that these ticks gained these bacterial agents from a common ancestor and/or by recent horizontal transmission of rickettsiae between ticks.     

Molecular Detection and Identification of Spotted Fever Group Rickettsiae in Ticks Collected from the West Bank,Palestinian Territories

Background

Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) rickettsiae. Although Spotted Fever is prevalent in the Middle East, no reports for the presence of tick-borne pathogens are available or any studies on the epidemiology of this disease in the West Bank. We aimed to identify the circulating hard tick vectors and genetically characterize SFG Rickettsia species in ixodid ticks from the West Bank-Palestinian territories.

Methodology/Principal Findings

A total of 1,123 ixodid ticks belonging to eight species (Haemaphysalis parva, Haemaphysalis adleri, Rhipicephalus turanicus, Rhipicephalus sanguineus, Rhipicephalus bursa, Hyalomma dromedarii, Hyalomma aegyptium and Hyalomma impeltatum) were collected from goats, sheep, camels, dogs, a wolf, a horse and a tortoise in different localities throughout the West Bank during the period of January-April, 2014. A total of 867 ticks were screened for the presence of rickettsiae by PCR targeting a partial sequence of the ompA gene followed by sequence analysis. Two additional genes, 17 kDa and 16SrRNA were also targeted for further characterization of the detected Rickettsia species. Rickettsial DNA was detected in 148 out of the 867 (17%) tested ticks. The infection rates in Rh. turanicus, Rh. sanguineus, H. adleri, H. parva, H. dromedarii, and H. impeltatum ticks were 41.7, 11.6, 16.7, 16.2, 11.8 and 20%, respectively. None of the ticks, belonging to the species Rh. bursa and H. aegyptium, were infected. Four SFG rickettsiae were identified: Rickettsia massiliae, Rickettsia africae, Candidatus Rickettsia barbariae and Candidatus Rickettsia goldwasserii.

Significance

The results of this study demonstrate the geographic distribution of SFG rickettsiae and clearly indicate the presence of at least four of them in collected ticks. Palestinian clinicians should be aware of emerging tick-borne diseases in the West Bank, particularly infections due to R. massiliae and R. africae.     

Assessment of Survival and Body Size Variation of Culicoides imicola (Diptera: Ceratopogonidae) as Functions of “Candidatus Cardinium” (Bacteroidetes) Infection Status

“Candidatus Cardinium hertigii” (Bacteroidetes) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of “Candidatus Cardinium” infection was documented as moderate, with no detectable sex bias. We therefore investigated whether “Candidatus Cardinium” affects important fitness parameters, such as survival and body size, in Culicoides imicola, a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account “Candidatus Cardinium” infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with “Candidatus Cardinium” infection data. The findings revealed no difference in survival of Culicoides infected with “Candidatus Cardinium” and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for “Candidatus Cardinium”-infected versus uninfected midges. In aggregate, these findings support our conclusion that “Candidatus Cardinium” does not have an overt effect on the survival and size of adult C. imicola midges. “Candidatus Cardinium” may affect immature stages or may alter adult reproductive performance.     

Single-cell genomics of a rare environmental alphaproteobacterium provides unique insights into Rickettsiaceae evolution

The bacterial family Rickettsiaceae includes a group of well-known etiological agents of many human and vertebrate diseases, including epidemic typhus-causing pathogen Rickettsia prowazekii. Owing to their medical relevance, rickettsiae have attracted a great deal of attention and their host-pathogen interactions have been thoroughly investigated. All known members display obligate intracellular lifestyles, and the best-studied genera, Rickettsia and Orientia, include species that are hosted by terrestrial arthropods. Their obligate intracellular lifestyle and host adaptation is reflected in the small size of their genomes, a general feature shared with all other families of the Rickettsiales. Yet, despite that the Rickettsiaceae and other Rickettsiales families have been extensively studied for decades, many details of the origin and evolution of their obligate host-association remain elusive. Here we report the discovery and single-cell sequencing of ‘Candidatus Arcanobacter lacustris'', a rare environmental alphaproteobacterium that was sampled from Damariscotta Lake that represents a deeply rooting sister lineage of the Rickettsiaceae. Intriguingly, phylogenomic and comparative analysis of the partial ‘Candidatus Arcanobacter lacustris'' genome revealed the presence chemotaxis genes and vertically inherited flagellar genes, a novelty in sequenced Rickettsiaceae, as well as several host-associated features. This finding suggests that the ancestor of the Rickettsiaceae might have had a facultative intracellular lifestyle. Our study underlines the efficacy of single-cell genomics for studying microbial diversity and evolution in general, and for rare microbial cells in particular.     

Characterization of the Denitrification-Associated Phosphorus Uptake Properties of “Candidatus Accumulibacter phosphatis” Clades in Sludge Subjected to Enhanced Biological Phosphorus Removal

To characterize the denitrifying phosphorus (P) uptake properties of “Candidatus Accumulibacter phosphatis,” a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of “Ca. Accumulibacter” and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence in situ hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized “Ca. Accumulibacter” subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (Dechloromonas [from 1.2% to 19.2%] and “Candidatus Competibacter phosphatis” [from 16.4% to 20.0%]), while the overall “Ca. Accumulibacter” abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the “Ca. Accumulibacter” clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/liter), all “Ca. Accumulibacter” clades successfully took up phosphorus in the presence of nitrate. However, the “Ca. Accumulibacter” clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by “Ca. Accumulibacter” clades occurred when nitrite was added. These results suggest that the “Ca. Accumulibacter” cells lack nitrate reduction capabilities and that P uptake by “Ca. Accumulibacter” is dependent upon nitrite generated by associated nitrate-reducing bacteria such as Dechloromonas and “Ca. Competibacter.”     

A Mesophilic,Autotrophic, Ammonia-Oxidizing Archaeon of Thaumarchaeal Group I.1a Cultivated from a Deep Oligotrophic Soil Horizon

Soil nitrification plays an important role in the reduction of soil fertility and in nitrate enrichment of groundwater. Various ammonia-oxidizing archaea (AOA) are considered to be members of the pool of ammonia-oxidizing microorganisms in soil. This study reports the discovery of a chemolithoautotrophic ammonia oxidizer that belongs to a distinct clade of nonmarine thaumarchaeal group I.1a, which is widespread in terrestrial environments. The archaeal strain MY2 was cultivated from a deep oligotrophic soil horizon. The similarity of the 16S rRNA gene sequence of strain MY2 to those of other cultivated group I.1a thaumarchaeota members, i.e., Nitrosopumilus maritimus and “Candidatus Nitrosoarchaeum koreensis,” is 92.9% for both species. Extensive growth assays showed that strain MY2 is chemolithoautotrophic, mesophilic (optimum temperature, 30°C), and neutrophilic (optimum pH, 7 to 7.5). The accumulation of nitrite above 1 mM inhibited ammonia oxidation, while ammonia oxidation itself was not inhibited in the presence of up to 5 mM ammonia. The genome size of strain MY2 was 1.76 Mb, similar to those of N. maritimus and “Ca. Nitrosoarchaeum koreensis,” and the repertoire of genes required for ammonia oxidation and carbon fixation in thaumarchaeal group I.1a was conserved. A high level of representation of conserved orthologous genes for signal transduction and motility in the noncore genome might be implicated in niche adaptation by strain MY2. On the basis of phenotypic, phylogenetic, and genomic characteristics, we propose the name “Candidatus Nitrosotenuis chungbukensis” for the ammonia-oxidizing archaeal strain MY2.     

Broad Distribution of Diverse Anaerobic Ammonium-Oxidizing Bacteria in Chinese Agricultural Soils

Anaerobic ammonium-oxidizing (anammox) bacteria have been detected in many marine and freshwater ecosystems. However, little is known about the distribution, diversity, and abundance of anammox bacteria in terrestrial ecosystems. In this study, anammox bacteria were found to be present in various agricultural soils collected from 32 different locations in China. Phylogenetic analysis of the 16S rRNA genes showed “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” in the collected soils, with “Candidatus Brocadia” being the dominant genus. Quantitative PCR showed that the abundance of anammox bacteria ranged from 6.38 × 10⁴ ± 0.42 × 10⁴ to 3.69 × 10⁶ ± 0.25 × 10⁶ copies per gram of dry weight. Different levels of diversity, composition, and abundance of the anammox bacterial communities were observed, and redundancy analysis indicated that the soil organic content and the distribution of anammox communities were correlated in the soils examined. Furthermore, Pearson correlation analysis showed that the diversity of the anammox bacteria was positively correlated with the soil ammonium content and the organic content, while the anammox bacterial abundance was positively correlated with the soil ammonium content. These results demonstrate the broad distribution of diverse anammox bacteria and its correlation with the soil environmental conditions within an extensive range of Chinese agricultural soils.     

A New Huanglongbing Species, “Candidatus Liberibacter psyllaurous,” Found To Infect Tomato and Potato, Is Vectored by the Psyllid Bactericera cockerelli (Sulc)

A new huanglongbing (HLB) “Candidatus Liberibacter” species is genetically characterized, and the bacterium is designated “Candidatus Liberibacter psyllaurous.” This bacterium infects the psyllid Bactericera cockerelli and its solanaceous host plants potato and tomato, potentially resulting in “psyllid yellowing.” Host plant-dependent HLB transmission and variation in psyllid infection frequencies are found.